The goal of this project is to understand how templated nucleotide polymerizing enzymes achieve fidelity. Previous workers have concentrated on the 3' yields 5' exonuclease associated with many DNA polymerases. However, the mechanisms by which the polymerizing step achieves accuracy have been largely ignored. To investigate the latter, the effect of polymerase substrate analogues on the fidelity of the polymerization reaction are being studied primarily using T4 DNA polymerase as a model sytem. Modified nucleotide triphosphates are being utilized as probes of fidelity mechanisms. Initial efforts are focusing on rigorously testing the assumed importance of classical Watson-Crick base pairing by utilizing substrates with altered H-bond configurations. Kinetic steps will be probed with substrate analogues modified in the triphosphate moiety.